Quantitation of hepcidin in human urine by liquid chromatography-mass spectrometry.
Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [15N,13C2]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine(-1) were found in healthy controls, with reduced levels in hemochromatosis (P<0.00006) and elevated levels in inflammation (P<0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P<0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.
Bansal
Sukhvinder S
SS
Pharmaceutical Sciences Division, King's College London, 150 Stamford Street, Waterloo, and Institute of Liver Studies, King's College Hospital, London SE1 9NH, UK. sukhi.bansal@kcl.ac.uk
Halket
John M
JM
Bomford
Adrian
A
Simpson
Robert J
RJ
Vasavda
Nisha
N
Thein
Swee Lay
SL
Hider
Robert C
RC
D000328:Adult; D000900:Anti-Bacterial Agents / Q000652:urine*; D023181:Antimicrobial Cationic Peptides / Q000652:urine*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D064451:Hepcidins; D006801:Humans; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins